GABAergic drugs and conflict behavior in the rat: Lack of similarities with the actions of benzodiazepines

Summary Effecs of drugs which enhance or reduce GABAergic neurotransmission upon conflict behavior were evaluated with a modified Vogel procedure which was shown to be insensitive to variations in motivation to drink and to the analgesic effects of morphine. In addition, the effects of these drugs on ambulatory activity and motor execution were quantified. For comparison, the benzodiazepines diazepam and chlordiazepoxide were used. Anticonflict actions of diazepam were obtained with a shock current of 0.25 mA but not with 0.05 or 0.5 mA, whereas the proconflict effect of FG7142 was obtained with 0.05 mA but not with higher currents. Diazepam and chlordiazepoxide had anxiolytic effect in a dose similar to that required to reduce ambulatory activity, but below that needed to affect motor execution. At doses high enough to impair motor execution, anticonflict effects were considerable. The GABA-A receptor agonist THIP and the GABA-B receptor agonist baclofen lacked effect on conflict behavior in moderate doses, which reduced ambulatory activity. In doses which produced motor deficiencies these drugs reduced licking both in the conflict test and when tested without shock administration. The effects of the GABA transaminase inhibitors 7-acetylen GABA and sodium valproate were similar to those of the receptor agonists. The GABA reuptake inhibitor SKF 100330A produced anticonflict effect in a dose below that needed to reduce ambulatory activity, but lacked effect on conflict behavior in higher doses. The GABA antagonist picrotoxin, and the GABA synthesis inhibitors 4-deoxypyridoxine and isoniazide, reduced licking both in the absence and presence of shock, and affected motor functions in the same doses. Bicuculline, at the doses used, had no behavioral effects. The different behavioral profiles of GABAergic agents and benzodiazepines, and the lack of consistent effects of the former on conflict behavior, seem to suggest that GABA receptors are not involved in conflict reduction. Further evidence for this hypothesis was obtained in experiments where it was found that the GABA antagonists bicuculline and picrotoxin did not block the effects of the benzodiazepines. No evidence was found for a tonic anticonflict action of GABAergic systems. It thus appears that anticonflict actions of benzodiazepines may be independent of GABAergic mechanisms.     

Schedule dependence of the interaction of naloxone and chlordiazepoxide.

Reports that the opiate antagonist, naloxone, blocks the anticonflict effects of diazepam and chlordiazepoxide suggest endogenous opioid involvement in the anxiolytic actions of the benzodiazepines. However, naloxone's ability to antagonize the anticonflict effects of the benzodiazepines is not universal, but schedule specific. The present experiments investigated the importance of the timing of conflict periods and control of reinforcement on the naloxone-benzodiazepine interaction. We tested the effects of naloxone (3 mg/kg, IP) and chlordiazepoxide (5 mg/kg, IP) on acquisition of a successive discrimination schedule, with nonreward periods similar in length and frequency to those of signalled DRL, and on an FI60-s schedule. Chlordiazepoxide increased rewarded responding and, unexpectedly, decreased nonrewarded responding during acquisition of successive discrimination. This reduction in nonrewarded responding was reversed by naloxone. Under the FI60 schedule, chlordiazepoxide increased nonrewarded responding, an effect that was totally blocked by naloxone at the beginning of the FI. Naloxone's ability to reverse the response-releasing effect of chlordiazepoxide decreased later in the FI. These results suggest endogenous opioid systems are involved in the anxiolytic actions of the benzodiazepines when the animal is adapting to recently introduced conflict. Once adaptation occurs, other neurotransmitter systems mediate the actions of the benzodiazepines.     

Naloxone potentiates anxiolytic-like actions of diazepam, pentobarbital and meprobamate but not those of Ro19-8022 in the rat

The elevated plus-maze test was used to determine if the opiate antagonist naloxone could potentiate the anxiolytic-like effects of the benzodiazepine diazepam, the barbiturate pentobarbital, the propanediol carbamate meprobamate and the partial benzodiazepine receptor agonist [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl) carbonyl]-2-pyrrolidine-methanol (Ro19-8022) in the rat. A subeffective dose of each of these compounds was combined with naloxone, 10 mg/kg. Naloxone had no effect by itself, but potentiated all drugs except Ro19-8022. The proportion of entries on the open arm increased while the total number of arms entries was not modified. These results coincide with and extend data previously obtained in the mouse. One possible explanation for naloxone's effect could be that it blocks opioid inhibition of GABAergic (gamma-aminobutyric acid) neurons thereby enhancing the effects of benzodiazepines. Another possibility is that naloxone blocks opioid effects on adenosinergic systems.     

Potentiation of the anticonflict effects of diazepam, but not pentobarbital and phenobarbital, by aminooxyacetic acid (AOAA)

The Conditioned Suppression of Drinking (CSD) paradigm is an "animal model" for anxiety which has been used to study the anticonflict effects of the benzodiazepines. It has been postulated that benzodiazepines produce their effects through interactions with GABA. The present study examined this potential GABA-BZ interaction on CSD behavior. In daily 10-minute sessions, water-deprived rats were trained to drink from a tube which was occasionally electrified (0.5 mA), electrification being signalled by a tone. Within 2-3 weeks control CSD responding had stabilized (16-24 shocks session and 10-14 ml water/session); drug tests were conducted at weekly intervals. As expected, diazepam (0.3-20.0 mg/kg), pentobarbital (0.6-10.0 mg/kg) and phenobarbital (10.0-40.0 mg/kg) alone markedly increased the number of shocks received at doses which did not depress background responding (i.e., water intake). Treatment with the GABA-transaminase inhibitor aminooxyacetic acid (AOAA: 2.5-10.0 mg/kg, 10- or 60-minute pretreatment) alone had no anticonflict effect on CSD behavior. However, pretreatment (60-minute) with 10.0 mg/kg AOAA significantly potentiated the effects of diazepam, as indicated by a significant shift to the left in the diazepam dose-response curve relative to diazepam alone. By contrast, the anticonflict effects of pentobarbital and phenobarbital were unaffected by this AOAA pretreatment. Thus, while increases in GABA transmission alone do not appear to affect CSD behavior, the anticonflict effect of benzodiazepines, but not barbiturates, appear to be potentiated by increases in GABA transmission.     

Restoration of shock-suppressed behavior by treatment with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801), a substance with potent anticonvulsant,central sympathomimetic,and apparent anxiolytic properties

MK-801 was evaluated in rats for “antipunishment” and “anticonflict” activity in two procedures: (1) A conditioned emotional response (CER) test involving the suppression of lever-pressing by unaviodable shock and (2) a simple conflict test in water-deprived animals that were shocked for licking water. The effect of MK-801 in both procedures was qualitatively similar to the benzodiazepines. Lever-pressing in the CER test was increased by MK-801 at doses ranging from 50–400 μg/kg administered orally (p.o.) at either 0.5 or 2 hours prior to testing. The number of shocks received in the “thirsty rat” conflict procedure was increased by MK-801 at doses from 110–1,000 μg/kg p.o., providing the compound was given 2 or more hours before test. MK-801 was without anticonflict activity when administered 1 hour prior to study. In squirrel monkeys trained in a response-contingent conflict paradigm, a specific anticonflict effect for MK-801 (50–400 μ/kg p.o.) was not demonstrable. As assessed by observing the overt behavior of squirrel monkeys, MK-801 at doses greater than 100 μg/kg p.o. caused apparent “taming” or “tranquilization.” Chlordizepoxide and diazepam given, respectively, at doses above 1 and 2 mg/kg p.o. had a similar “taming” action. The benzodiazepines possessed a greater separation between doses producing “taming” and those causing ataxia than did MK-801. The mode of action for the antipunishment effect of MK-801 in rats is not known, but it was found that naloxone (2 or 5 mg/kg SC) antagonized the anticonflict actions of both MK-801 and chlordiazepoxide. In vitro, MK-801 was inactive (IC₅₀ > 2 μM) with respect to competing for binding to rat brain tissue by various radioligands (diazepam, muscimol, apomorphine, spiroperidol, serotonin, LSD, WB-4101, dihydroalprenolol, QNB, and 2-chloroadenosine). An increase in ³H-diazepam binding in vitro in rat brain tissue was detected following acute, but not chronic, treatment in vivo with MK-801.     

6-(Alkylamino)-3-aryl-1,2,4-triazolo[3,4-a]phthalazines. A new class of benzodiazepine receptor ligands

Some 6-(alkylamino)-3-aryl-1,2,4-triazolo[3,4-a]phthalazines have been shown to displace diazepam from rat brain specific binding sites, in vitro, with Ki (nM) values comparable to those of reference benzodiazepines and to have anticonvulsant (pentylenetetrazole test, mice) and anticonflict activity (Vogel test, rat) in vivo. Separation between the doses causing anticonflict effects (Vogel test, rat) and those impairing motor coordination (rotarod test, rat) has been shown for N,N-bis(2-methoxyethyl)-3-(4-methoxyphenyl)-1,2,4-triazolo[3,4-a] phthalazin-6-amine (80). This compound, unlike diazepam, was inactive in counteracting the strychnine (mouse) and maximal electroshock (mouse) induced convulsions and in the "aggressive monkey" model. These differences from the classical benzodiazepines in the animal tests indicate that 80 may have some selective anxiolytic activity.     

Naloxone blocks the effect of diazepam and meprobamate on conflict behaviour in rats

The effect of naloxone on the anticonflict action of diazepam was studied in a model involving foot shock-induced suppression of food-rewarded operant behaviour. Both 1 and 10 mg/kg naloxone SC abolished the increase in punished responding produced by diazepam and chlordiazepoxide. Naloxone also blocked the anticonflict effect of meprobamate. These observations are discussed in terms of a possible involvement of endogenous opioid peptides in the anxiolytic effects of tranquillizers.     

Midazolam-induced rapid changes in licking behaviour: evidence for involvement of endogenous opioid peptides

The role of endogenous opioid peptides in the effects of midazolam on ingestive behaviour was investigated using a detailed analysis of licking behaviour in the rat. Midazolam (1.8 mg/kg IP) was administered in combination with either flumazenil (10 and 20 mg/kg IP) or naloxone (0.1 and 0.3 mg/kg IP). The effect on licking patterns during 60-s exposures to a range of concentrations of a fat emulsion (Intralipid) was then recorded. Midazolam significantly increased the total number of licks for Intralipid by increasing the mean bout duration. This effect is consistent with the proposal that benzodiazepines enhance palatability. Flumazenil and naloxone were ineffective when administered alone, but both drugs blocked the effect of midazolam on total number of licks by selectively attenuating mean bout duration. Midazolam also produced a significant decrease in the intrabout lick rate, probably due to the muscle relaxant effects of this drug. This decrease in the intrabout lick rate was reversed by pretreatment with flumazenil but not by naloxone. The results suggest that endogenous opioids may be important for the palatability effects of midazolam, but may not be involved in the muscle relaxant effects of this drug. Received: 20 August 1996/Final version: 16 January 1997     

Interactions between opioids and cocaine on locomotor activity in rats: influence of an opioid's relative efficacy at the mu receptor

Rationale. Cocaine and mu opioid agonists increase central dopamine concentrations and produce robust interactions at both neurochemical and behavioral levels. Although the interactions between cocaine and high-efficacy mu opioids have been well characterized, the interactions between cocaine and lower efficacy opioids have not been as extensively examined. Objective. The purpose of this study was to examine the interactions between cocaine and opioids possessing a range of relative efficacy at the mu receptor. Methods. Male, Long-Evans rats were habituated to an open-field, locomotor activity chamber, and the effects of cocaine and various opioids were tested under a cumulative dosing procedure. In this procedure, a selected dose of an opioid was administered during the first component of a session, with increasing doses of cocaine administered during subsequent components. Results. When administered alone, cocaine produced dose-dependent increases in locomotor activity that was stable across 5 weeks of behavioral testing. The high-efficacy mu opioid levorphanol, and the low-efficacy opioids buprenorphine, butorphanol, nalbuphine and (−)-pentazocine, dose-dependently enhanced the effects of cocaine at doses that did not alter locomotor activity when administered alone. In contrast, the opioid antagonist naloxone, and to a lesser extent, the kappa opioid spiradoline attenuated the effects of cocaine at doses that did not alter locomotor activity when administered alone. Across an extensive dose range, the low-efficacy opioid nalorphine failed to alter cocaine's locomotor-activating effects. Conclusions. These data suggest that low-efficacy opioids possessing significant mu-agonist activity (e.g. buprenorphine, butorphanol, nalbuphine, (−)-pentazocine) may potentiate the effects of cocaine in a manner similar to that typically observed with high-efficacy mu opioids. Electronic Publication     

Acute administration of diazepam and buspirone in rats trained on conflict schedules having different degrees of predictability

The anti-conflict activities of diazepam and buspirone were examined on three schedules designed to condition the suppression of licking. The schedules differed in the degree to which they predicted (signalled) the presentation of a conflict inducing electric shock. The first study investigated the effects of three doses of diazepam (0.5, 2, and 5 mg/kg IP) on a predictable, a moderately predictable, and an unpredictable schedule of shock presentation. Diazepam induced a significant increase from baseline in licking during the shock component on all three schedules. These anticonflict effects were the most consistent on the predictable schedule, and least consistent on the unpredictable schedule. A second experiment investigated the anticonflict activity of three doses of buspirone (0.125, 0.25, and 0.625 mg/kg SC) on each of these three schedules. The predictable and moderately predictable schedules failed to detect anticonflict activity at any dose of buspirone. However, the lowest dose (0.125 mg/kg) of buspirone increased shocked licking and the highest dose (0.625 mg/kg) decreased shock component licking on the unpredictable schedule. Thus the unpredictable schedule was sensitive to both anticonflict (anxiolytic) and proconflict (anxiogenic) effects of buspirone.     

Effects of diazepam on conflict behaviour and on plasma corticosterone levels in male and female rats

The anxiolytic properties of diazepam and its effects on plasma corticosterone levels were compared in male and female, water deprived rats exposed to the punished (0.8 mA) drinking procedure. The effects of diazepam on unpunished licking, tested under familiar or unfamiliar conditions, and on the lick latency were also studied and a comparison between the two sexes was made. Both punished and unpunished drinking were less in females than in males. In both sexes, a clear anticonflict effect, i.e. a much greater effect on punished than on unpunished drinking, was obtained with 2 and 4 mg/kg, but not with I mg/kg, of diazepam i.p. Plasma corticosterone levels were higher in water deprived females than in males. Following the punished and unpunished drinking procedure, plasma corticosterone levels were found to have decreased more in female than in male rats, especially after administration of 1 mg/kg of diazepam. Diazepam had similar anticonflict effects in rats of both sexes but had a greater suppressive effect on the plasma corticosterone levels in female rats. There was no correlation between the anxiolytic effects of diazepam and its effect on the plasma corticosterone levels. When testing was done under unfamiliar conditions, the latency to licking was greater in female than in male rats and diazepam (1, 2 and 4 mg/kg) increased this latency in both sexes. The results suggest sex differences in the neuroendocrine, but not in the anxiolytic, effects of diazepam.     

Emetic and antiemetic effects of opioids in the dog

The emetic and antiemetic effects of opioid agonists were studied in awake dogs. The mu-agonists morphine, fentanyl and methadone, in sedative doses, prevented the emetic response to apomorphine and copper sulphate; only morphine induced emesis, at doses lower than those required to prevent emesis. The delta-agonist [D-Ala2,Met5]enkephalinamide (DALA) and [Leu5]enkephalin induced emesis in some of the dogs studied but had no antiemetic activity. The kappa-agonists bremazocine and ethylketocyclazocine (EKC) did not induce emesis but, at sedative doses, prevented the emetic response to apomorphine. The emetic effect of DALA was antagonized by naloxone in some dogs; the antiemetic effect of morphine, bremazocine and EKC was blocked by both naloxone and MR 2266. The non-opioid sedatives diazepam, phenobarbital and xylazine, administered in sedative doses, did not prevent apomorphine-induced emesis. Our results suggest that a delta-receptor is involved in the emetic effect and a mu- and/or or kappa-receptor in the antiemetic effect of opioids.     

Effects of psychoactive drugs in the Vogel conflict test in mice.

This study examined effects of various psychoactive drugs on the Vogel conflict test, where drinking behavior is punished by electric shocks, in ICR mice to clarify the pharmacological features of this method in mice. A benzodiazepine anxiolytic diazepam and a barbiturate pentobarbital produced significant anticonflict effects, which mean that these drugs increased the number of electric shocks mice received during 40-min test session. On the other hand, yohimbine (alpha2-receptor antagonist), caffeine (adenosine-receptor antagonist), scopolamine (muscarinic cholinergic antagonist), cyclazocine (sigma-receptor antagonist), cimetidine (H2-receptor antagonist), baclofen (GABA(B)-receptor agonist), MK-801 (NMDA-receptor antagonist), buspirone (5-HT1A-receptor agonist), chlorpromazine (dopamine-receptor antagonist) and haloperidol (dopamine-receptor and sigma-receptor antagonist) all did not produce anticonflict effects in this test using ICR mice. The results suggest that the Vogel conflict test is applicable to ICR mice and that this test in mice is appropriate as a screening method for drugs that have apparent anti-anxiety actions.     

Discriminative stimulus effects of tizanidine hydrochloride: Studies with rats trained to discriminate either tizanidine,clonidine, diazepam,fentanyl, or cocaine

Male Sprague Dawley rats were trained in a two-lever food-reinforced procedure to discriminate between the effects of saline and either tizanidine hydrochloride, clonidine hydrochloride, diazepam, fentanyl, or cocaine hydrochloride. Tizanidine-trained rats dose-dependently generalized the effects of tizanidine and clonidine but not pentobarbital, diazepam, morphine, or cocaine. Clonidine-trained rats dose-dependently generalized the effects of clonidine and tizanidine but not pentobarbital, diazepam, or morphine. Diazepam-trained rats dose-dependently generalized the effects of diazepam but did not generalize tizanidine. Fentanyl-trained rats dose-dependently generalized the effects of fentanyl but did not generalize tizanidine. Cocaine-trained rats did not generalize the effects of tizanidine to the cocaine discriminative stimulus. Yohimbine hydrochloride but not naloxone hydrochloride dose-dependently antagonized the discriminative stimuli produced by both tizanidine and clonidine. These data demonstrate that tizanidine shares discriminative stimulus properties with clonidine but not with pentobarbital, diazepam, fentanyl, morphine, or cocaine. The discriminative stimuli produced by tizanidine and clonidine are mediated via an agonistic interaction with alpha₂-adrenergic receptors and not via an agonistic interaction with opioid receptors.     

Naloxone and chlordiazepoxide: effects on acquisition and performance of signalled punishment

The opiate antagonist, naloxone, has been reported to attenuate the loss of behavioural inhibition produced by benzodiazepines in tasks involving punishment and nonreward. This has led to suggestions that endogenous opioid systems may be involved in the anxiolytic actions of the benzodiazepines. However, the capacity of naloxone to block the effects of benzodiazepines depends on the behavioural schedule used. We tested the effects of naloxone and chlordiazepoxide on acquisition and performance of a signalled punishment schedule. Chlordiazepoxide (5 mg/kg IP) increased both punished and unpunished responding during acquisition and unpunished responding during performance of the conflict schedule. Naloxone (3 mg/kg IP) was essentially without effect on responding and failed to attenuate the punishment-releasing effects of chlordiazepoxide. The failure of naloxone and chlordiazepoxide to interact during acquisition of this punishment schedule is similar to results we obtained with successive discrimination and is in contrast to our findings with a differential reinforcement of low rates schedule. These results are consistent with the view that benzodiazepines reduce behavioural inhibition through two separate routes; that one of these routes depends on the release of endogenous opiates; and that the predominant route depends on schedule parameters.     

The effect of opiates and naloxone on food intake in virgin and lactating rats

Lactation provides an excellent model of non-obese hyperphagia. There is accumulating evidence that endogenous opioids play a role in the modulation of the hormonal changes that occur during lactation. Because endogenous opioids appear also to play a role in the regulation of feeding, we studied the effects of the opiate agonist, butorphanol tartrate, and an opiate antagonist, naloxone, on food intake in virgin female rats and in rats during early, mid and late lactation and during post-weaning. It has been reported that female rats are less sensitive to the suppressant effects of nalmefene, an opioid antagonist, than male rats. Therefore, we also examined the effect of naloxone, an opioid antagonist, on spontaneous nocturnal feeding and 24 hour food deprivation-induced food intake in virgin female rats. We found that female rats were relatively insensitive to the food suppressant effects of naloxone following 24 hour food deprivation, while male rats tested under similar conditions had a decreased intake in response to naloxone. Despite the marked hyperphagia that occurred during lactation, there were minimal alterations in the response to opiate agonists and antagonists during this time period. Our data suggest that endogenous opioids may not play a pivotal role in the hyperphagia of lactation.     

Reversal of antinociceptive effect of cholecystokinin by benzodiazepines and a benzodiazepine antagonist, Ro 15-1788

Intraperitoneally administered benzodiazepines, chlordiazepoxide (2-5 mg/kg), diazepam (1 mg/kg), flurazepam (1 mg/kg) and a benzodiazepine antagonist, Ro 15-1788 (0.5 mg/kg), reversed the antinociceptive effect in mice which was induced by intracisternal administration of 1 microgram of sulfated cholecystokinin octapeptide. The antinociceptive effect of cholecystokinin was reversed by naloxone, suggesting that the antinociceptive action involves endogenous opioid peptides in its production. On the other hand, morphine-induced analgesia was not reversed by diazepam and Ro 15-1788. These facts rule out opioid receptors as the site of the antagonism between the benzodiazepines or Ro 15-1788 and cholecystokinin on the antinociceptive effect. Benzodiazepines and Ro 15-1788 seem to inhibit the release of opioid peptides induced by cholecystokinin.     

Quaternary opiate antagonists lower blood pressure and inhibit leucine-enkephalin responses

The quaternary opiate antagonists naloxone methylbromide (MB) and naltrexone MB do not cross the blood-brain barrier, and may be used to differentiate peripheral from central nervous system effects of the opioid peptides. When administered intravenously to the conscious, chronically instrumented dog, naloxone MB transiently reduces mean systemic arterial pressure and increases heart rate in a dose-dependent manner over the concentration range from 0.1 to 1.0 mg/kg. The [Leu5]enkephalin response is completely inhibited at naloxone MB doses as low as 0.25 mg/kg, but this inhibition has terminated by 30 min after dosing. Naltrexone MB displays a similar spectrum of activity. This inhibition of the intravenous [Leu5]enkephalin response by the quaternary opiate antagonists indicates that the [Leu5]enkephalin response occurs by activation of peripheral receptor sites. The decrease in mean pressure induced by these antagonists coupled with the inhibition of the [Leu5]enkephalin response suggests that peripheral enkephalins may play a role in blood pressure regulation.     

Naloxone in the prevention of the adverse cognitive effects of ECT: a within-subject, placebo controlled study.

Electroconvulsive therapy (ECT) is a highly effective treatment for major depression, but is also associated with characteristic cognitive side effects. Several reports document that endogenous opioids and their receptors are activated by electroconvulsive shock (ECS) and that naloxone in doses sufficient to block endogenous opioid receptors may reverse ECS-induced retrograde amnesia. This placebo-controlled, randomized, within-patient study was conducted to examine the potential of naloxone, given in doses sufficient to block opioid receptors (high dose), to ameliorate acute anterograde and retrograde memory impairments following ECT. Compared to placebo and low dose naloxone, high dose naloxone administered immediately before ECT resulted in significant reductions in anterograde amnesia, and better performance on an attention task. Both low and high dose naloxone improved verbal fluency. There were no beneficial effects of high dose naloxone on retrograde amnesia, and an indication that high dose naloxone may have worsened retrograde amnesia for shape stimuli. There were no effects of high dose naloxone on seizure duration, vital signs, and subjective side effects. The study is consistent with prior research in which change in behavioral and physiological measures was produced principally by naloxone doses sufficient to block endogenous opioid receptors and offers evidence of the potential for ameliorating some adverse cognitive effects associated with ECT.     

Pharmacologic characterization of a novel non-benzodiazepine selective anxiolytic, DN-2327

DN-2327, 2-(7-chloro-1,8-naphthyridin-2-yl)-3-[(1,4-dioxa-8- azaspiro[4.5]dec-8-yl)carbonylmethyl]isoindolin-1-one, produced anxiolytic, taming and anti-convulsive effects when administered orally to several species of animals. DN-2327 produced few of the sedative-hypnotic and muscle-relaxant effects observed with diazepam. The durations of the anxiolytic and anti-convulsive activities of DN-2327 were much longer than those of diazepam. Tolerance to DN-2327 did not develop when it was administered daily for 14 days in an anti-conflict test (Vogel conflict test). DN-2327 showed potent displacement activity against [3H]diazepam binding. The binding affinity of DN-2327 for benzodiazepine receptors was about twenty times that of diazepam. Furthermore, the affinity of DN-2327 for benzodiazepine receptors was not enhanced by the presence of GABA. There is a wide margin between the doses of DN-2327 that cause the anxiolytic effects and its sedative-hypnotic/muscle-relaxant effects. These results suggest that DN-2327 has more marked anxioselective properties compared with the benzodiazepines.